(a) Field of the Invention
The invention relates to the use of Saccharomyces cerevisiae highly related mannosyltransferases encoding genes that, when inhibited, cause lethality of the yeast cell and to a novel cell-based antifungal screening assay.
(b) Description of Prior Art
Fungi constitute a vital part of our ecosystem but once they penetrate the human body and start spreading they cause an infection or "mycosis" and they can pose a serious threat to human health. Fungal infections have dramatically increased in the last two (2) decades with the development of more sophisticated medical interventions and are becoming a significant cause of morbidity and mortality. Infections due to pathogenic fungi are frequently acquired by debilitated patients with depressed cell-mediated immunity such as those with HIV and now also constitute a common complication of many medical and surgical therapies. Risk factors that predispose individuals to the development of mycosis include neutropenia, use of immunosuppressive agents at the time of organ transplants, intensive chemotherapy and irradiation for hematopoietic malignancies or solid tumors, use of corticosteroids, extensive surgery and prosthetic devices, indwelling venous catheters, hyperalimentation and intravenous drug use, and when the delicate balance of the normal flora is altered through antimicrobial therapy.
The yeast genus Candida constitutes one of the major groups that cause systemic fungal infections and the five medically relevant species which are most often recovered from patients are C. albicans, C. tropicalis, C. glabrata, C. parapsilosis and C. krusei.
Much of the structure of fungal and animal cells along with their physiology and metabolism is highly conserved. This conservation in cellular function has made it difficult to find agents that selectively discriminate between pathogenic fungi and their human hosts, in the way that antibiotics do between bacteria and man. Because of this, the common antifungal drugs, like amphotericin B and the azole-based compounds are often of limited efficacy and are frequently highly toxic. In spite of these drawbacks, early initiation of antifungal therapy is crucial in increasing the survival rate of patients with disseminated candidiasis. Moreover, resistance to antifungal drugs is becoming more and more prominent. For example, 6 years after the introduction of fluconazole, an alarming proportion of Candida strains isolated from infected patients have been found to be resistant to this drug and this is especially the case with vaginal infections. There is thus, a real and urgent need for specific antifungal drugs to treat mycosis.
The fungal cell wall: a resource for new antifungal targets
In recent years, we have focused our attention on the fungal extracellular matrix, where the cell wall constitutes an essential, fungi-specific organelle that is absent from human/mammalian cells, and hence offers an excellent potential target for specific antifungal antibiotics. The cell wall of fungi is essential not only in maintaining the osmotic integrity of the fungal cell but also in cell growth, division and morphology. The cell wall contains a range of polysaccharide polymers, including chitin, .beta.-glucans and O-Serine/Threonine-linked mannose sidechains of glycoproteins. .beta.-glucans, homopolymers of glucose, are the main structural component of yeast cell wall, and constitute up to 60% of the dry weight of the cell wall. Based on their chemical linkage, two different types of polymers can be found: .beta.1,3-glucan and .beta.1,6-glucan.
Mannoproteins are an intrinsic part of the cell wall where they are intercalated in the meshwork of the glucose and chitin polymers and the attachment of mannose to cell surface proteins is a process essential for fungal viability. A great variety of cell surface mannoproteins are cross-linked through disulfide bounds or linked to cell wall polymers through glycosidic bonds. Many of these cell wall glycoproteins are of unknown function and do not appear to possess any enzymatic activity, but likely have structural roles in cell wall architecture and integrity.
It would be highly desirable to be provided with the identification and subsequent validation of new cell wall related targets that can be used in specific enzymatic and cellular assays leading to the discovery of new clinically useful antifungal compounds.